Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.
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PMID:Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol. 174 91

Our previous study suggested that N,N-dimethylsphingosine, but not unsubstituted sphingosine, could be a modulator of protein kinase C in epidermoid carcinoma A431 cells, since N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial sphingosine preparation has an enhancing effect on epidermal growth factor (EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of N,N-dimethylsphingosine as compared with lyso-glycosphingolipids and other sphingolipid breakdown products on EGF receptor autophosphorylation and (ii) demonstration of endogenous N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted sphingosine in A431 cells. The autophosphorylation of EGF receptor in the absence of detergent was strongly enhanced by N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of EGF and synergistic in the presence of EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-glycosphingolipids tested. Labeling of sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of sphingolipids by standard procedures, resulted in clear bands corresponding to N,N-dimethylsphingosine and ceramide, whereas the band corresponding to sphingosine was virtually absent. The bands corresponding to N,N-dimethylsphingosine and ceramide intensified when cells were treated with metabolic inhibitor for UDP-Glc:Cer beta-Glc transferase (which causes accumulation of ceramide). These results indicate that N,N-dimethylsphingosine acts as a stereospecific enhancer for EGF receptor kinase and is able to produce EGF-like activity in vitro even in the absence of EGF and detergent. Under physiological conditions, N,N-dimethylsphingosine is the major catabolite resulting from ceramide breakdown.
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PMID:A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells. 231 19

This report presents the first demonstration of a ceramide-hydrolyzing activity in mammalian epidermis. An assay using fractions derived from porcine epidermis and synthetic [3H]ceramide is described, and it is shown that under the conditions used, the Km for ceramide is 110 microM and hydrolysis is linear for up to 2 h. The enzyme activity is maximal at pH 8-9. The specific activity of ceramide hydrolase decreases as the protein concentration in the assay mixture increases, suggesting the possibility of a dissociable inhibitor. Also, the activity can be inhibited by added palmitic acid. Ceramide hydrolysis may be an important regulatory mechanism in the epidermis due to the ability of the liberated free sphingosine to modulate the activity of protein kinase C.
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PMID:Ceramidase activity in porcine epidermis. 238 45

Ceramide, a product arising from sphingomyelinase activity, has been shown to act as an intracellular second messenger in effecting growth inhibition, cellular differentiation, and apoptosis. In the present study, the relative effects of cell-permeable ceramides, N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide), on neutrophil responses were measured. When cells were activated with fMet-Leu-Phe, C2-ceramide both potentiated (< 1 microM) and inhibited (> 1 microM) superoxide generation. C2- and C6-ceramide inhibited phorbol ester-induced superoxide release from neutrophils at IC50 values of 5 and 120 microM, respectively. C2-ceramide had no effect on semipurified protein kinase C activity. Neither ceramide affected significantly the general level of phosphorylated proteins in phorbol ester-treated cells. C2-ceramide (1-20 microM) alone did not change cytosolic free Ca2+ levels but inhibited Ca2+ and Mn2+ influx in fMet-Leu-Phe-activated neutrophils. In contrast, sphingosine enhanced Ca2+ entry; thus, ceramide conversion to sphingosine was not significant. Unlike C2-ceramide, C2-dihydroceramide failed to block superoxide generation or Ca2+ influx. Preincubation of cells with 10 nM okadaic acid reversed slightly the effects of C2-ceramide. Calyculin A, tautomycin, and much higher concentrations of okadaic acid inhibited agonist-induced Ca2+ influx. We postulate that C2-ceramide may inhibit neutrophil superoxide release by activation of type 2A protein phosphatases. Results suggest that protein phosphatase type 1 up-regulates Ca2+ entry, whereas type 2A (or a ceramide-activated subtype) forestalls Ca2+ entry by inactivating a calcium influx factor.
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PMID:N-acetylsphingosine (C2-ceramide) inhibited neutrophil superoxide formation and calcium influx. 785 86

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.
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PMID:Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D. 789 Jun 7

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are cytokines with pleiotropic biological activities, exerting a broad range of overlapping biological functions. The redundancy of TNF and IL-1 activities may be based on the utilization of shared key components of intracellular signaling pathways. Two lipid second messengers have been found to transmit TNF and IL-1 intracellular signals: 1,2-diacylglycerol (DAG), generated by a phosphatidylcholine-specific phospholipase C, and ceramide, generated by sphingomyelinase (SMase). DAG is a well established activator of the important signaling system protein kinase C (PKC), which appears to mediate various cellular responses to TNF or IL-1. In addition, it is obvious that DAG also activates other enzyme systems like acidic sphingomyelinase. SMases have been implicated in a number of TNF responses, including stimulation of cell growth and differentiation, as well as triggering cytotoxicity and apoptosis. The metabolic active cleavage product of SMase, ceramide, is a novel multifunctional lipid second messenger capable of inducing various signaling systems. Both cytokines, TNF and IL-1, stimulate a neutral,plasma membrane-associated SMase that leads to stimulation of a protein kinase and eventually to activation of the mitogen-activated protein (MAP) kinase cascade and phospholipase A2. Ceramide is also capable of stimulating a cytosolic protein phosphatase. PKC plays a role in activation of the nuclear transcription factor AP-1, and the DAG-regulated acidic SMase is involved in transducing TNF signals to the cell nucleus via activation of the nuclear transcription factor NF-kappa B.
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PMID:The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. 796 60

Treatment of human neutrophils with tumor necrosis factor-alpha (TNF-alpha) resulted in an increase in concentration of ceramide and its catabolite, sphingosine. Sphingosine, a potent endogenous protein kinase C (PKC) inhibitor, as well as TNF-alpha, induced internucleosomal DNA fragmentation and morphological changes characteristic of apoptotic cells. Ceramide and sphingosine-1-phosphate, the initial product of sphingosine catabolism, did not cause apoptosis under our experimental conditions. In addition, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and N,N-dimethylsphingosine (DMS), known as PKC inhibitors, also induced apoptosis, suggesting that induction of apoptosis by sphingosine may be related to inhibition of PKC activity. These results indicate that sphingosine deacylated from ceramide may be an endogenous modulator mediating apoptotic signals by TNF-alpha in neutrophils.
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PMID:A possible role of sphingosine in induction of apoptosis by tumor necrosis factor-alpha in human neutrophils. 798 86

Prior studies demonstrated that increased intracellular availability of ceramide induces apoptotic DNA degradation and cell death in the human leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77). The present findings show that diglyceride opposes ceramide-related apoptosis in HL-60 and U937 cells. Acute (6-12-h) exposure to sphingomyelinase (100 milliunits/ml) or synthetic ceramide (10 microM) promoted apoptotic degradation of genomic DNA as indicated by (a) the appearance of both approximately 50-kilobase pair (kbp) DNA fragments and approximately 0.2-1.2-kbp DNA fragment ladders on agarose gels, (b) formation and release of small double-stranded DNA fragments, and (c) loss of integrity of bulk DNA. DNA damage was associated with reduced clonogenicity and expression of apoptotic morphology. In contrast, exposure to phospholipase C (0.001-100 milliunits/ml) or synthetic diglyceride (10 microM) failed to promote apoptosis and abolished the lethal actions of ceramide as defined by each of the indices outlined above. Ceramide-related apoptosis was also reduced by acute (6-h) exposure to tumor promoters such as phorbol dibutyrate and mezerein and the non-tumor-promoting agent bryostatin 1; conversely, chronic (24-h) pretreatment with these agents failed to modify ceramide-mediated cytotoxicity, but abolished the protective actions of diglyceride. These findings demonstrate that diglyceride and pharmacological protein kinase C activators reduce or abolish ceramide-mediated apoptosis in human leukemia cells and support the concept of a cytoprotective function for protein kinase C in the regulation of leukemic cell survival. In addition, the capacity of diglyceride to prevent very early genomic lesions (e.g. generation of 50-kbp DNA fragments) suggests that acute activation of protein kinase C arrests apoptosis at an initial stage.
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PMID:Attenuation of ceramide-induced apoptosis by diglyceride in human myeloid leukemia cells. 798 41

The cytokine-mediated stimulation of sphingomyelin (SM) metabolism is emerging as an important signal transduction pathway via the generation of ceramide and sphingosine, products which have been shown to affect a wide variety of biological processes. Because SM-mediated signal transduction is initiated via the hydrolysis of an integral membrane phospholipid by a phospholipase C-like enzyme (sphingomyelinase) to yield lipids which modulate protein kinase C activity, the SM and phosphatidylinositol (PI) signaling pathways share certain similarities. The present study was undertaken to examine the potential for interplay between SM and PI turnover by testing the effects of sphingosine, sphingosine-1-phosphate, and ceramide on PI turnover. In dermal fibroblasts, sphingosine stimulated a rapid dose-dependent hydrolysis of PI, yielding inositol 1,4,5-triphosphate, followed by increased levels of intracellular calcium. Sphingosine-induced inositol phosphate (IP) accumulation was observed between 5 and 30 microM sphingosine with a maximal accumulation of 2.7-fold over control levels. Enhanced IP formation was measured as early as 5 s following sphingosine treatment and IP levels remained elevated for more than 60 min. Intracellular calcium mobilization accompanied the dose-dependent accumulation of IPs in response to sphingosine, although this effect was not apparent until after a 30-40-s lag period. Interestingly, sphingosine-1-phosphate stimulated a more rapid release of intracellular Ca2+ than sphingosine, but it had no effect on PI turnover. DL-threo-Dihydrosphingosine, a competitive inhibitor of sphingosine kinase, stimulates both PI turnover and Ca2+ flux, but does not block the action of sphingosine relative to those two processes. Ceramide (added as C2-ceramide), N-stearylamine, and stearoyl-D-sphingosine did not affect PI turnover or Ca2+ mobilization. Pretreatment of intact cells with pertussis toxin partially inhibited sphingosine-mediated IP accumulation, suggesting a role for guanine nucleotide binding protein(s) (G protein) in sphingosine-stimulated PI turnover. Furthermore, guanosine 5'-O-(3-thiotriphosphate) stimulated, whereas guanosine 5'-O-(2-thiodiphosphate) inhibited, sphingosine-induced IP accumulation in permeabilized cells. Collectively, these data suggest that sphingosine enhances PI turnover by stimulating phospholipase C activity, and the activation of this process may be modulated by G protein interactions. Thus, the regulation of PI turnover and Ca2+ mobilization by sphingosine may represent another mechanism by which sphingosine modulates cell function and that these effects can be distinguished from those of ceramide.
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PMID:Sphingosine-mediated phosphatidylinositol metabolism and calcium mobilization. 811 27

The sphingolipids are a family of lipids found ubiquitously in eukaryotic cell membranes. Within the last decade sphingolipids have emerged as active participants in the regulation of cell growth, differentiation, transformation, and cell-cell contact. A prototypic sphingolipid signalling pathway is the 'sphingomyelin cycle,' in which membrane sphingomyelin is hydrolyzed in response to extracellular stimuli, generating the putative second messenger ceramide. Ceramide, in turn, is thought to propagate the signal into the cell interior by the activation of a phosphatase. It is likely that other sphingolipids are components of similar signalling cycles, generating a variety of lipid messengers which participate in as yet undefined pathways. Sphingosine, for example, is a potential breakdown product of all sphingolipids, and is well-known for its pharmacologic inhibition of protein kinase C. However, it is becoming apparent that sphingosine is active in multiple signalling cascades that are independent of protein kinase C, including effects on fibroblast cell growth and the regulation of the retinoblastoma tumor suppressor protein. Similarly, lyso-sphingolipids, while comprising only a minor fraction of the cell's total sphingolipids, are turning out to have biological effects which warrant their investigation as potential signalling molecules. A distinguishing characteristic of sphingolipid breakdown products is their apparent participation in anti-proliferative pathways of cell regulation. Thus, sphingolipid breakdown products can be found to play roles in growth inhibition, induction of differentiation, and programmed cell death. In coordination with other cellular signal transduction pathways, the sphingolipid breakdown products may be the harnesses on cell growth and may also contribute to the suppression of oncogenesis.
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PMID:Sphingolipid breakdown products: anti-proliferative and tumor-suppressor lipids. 828 Jul 42


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